Lactobacillus-based  ecoli  inhibition

ABSTRACT

A method of reducing growth of pathogenic bacteria in a gastrointestinal tract of an animal by providing to the animal a composition having 1×10 13  cfu of  Lactobacillus  probiotic microorganisms per kilogram of feed. When 1×10 4  viable cells of the  Lactobacillus  probiotic are added to 1×10 4  viable cells of  Escherichia coli  in a solution of MRS broth in a test tube and incubated in a water bath at 37° C. for six hours, the growth of  Escherichia coli  is reduced compared to a preparation of 1×10 4  viable cells of  Escherichia coli  mixed in a solution of MRS broth in a test tube and incubated in a water bath at 37° C. for six hours without the addition of  Lactobacillus  probiotic.

This application is a continuation of U.S. patent application Ser. No.15/351,134 filed Nov. 14, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/019,170 filed on Feb. 1, 2011, issued as U.S.Pat. No. 9,492,487 on Nov. 15, 2016 and claims the benefit of U.S.Provisional Patent Application 61/300,294 filed on Feb. 1, 2010, eachapplication of which is hereby expressly incorporated herein byreference in its entirety, without disclaimer.

BACKGROUND a. Field

The present disclosure relates generally to compositions and methods formanufacture and use of a multiple strain containing microbial product.More specifically, the disclosure relates to methods for improvinganimal health and/or productivity through the use of probioticmicroorganisms.

b. Description of the Related Art

One of the larger economic burdens facing dairy farmers is the high costof rearing and/or replacing heifers to maintain or increase herd size. Amajor factor contributing to the high cost of heifer replacement is theprevalence of diarrheal disease, known as scours, in livestock. Scourscauses greater than 60% of all deaths associated with pre-weaned calves,and accounts for 6.2% of total calf losses. The prevalence of scours canvary dramatically (4.3% to 52.4%) depending on herd, diet, season, or“outbreak” occurrences. Estimates of scouring rates within a herd aredifficult to obtain, though they are believed to between 15% and 35%.Nonetheless, it is agreed that diarrheal events comprise the largesthealth challenge to pre-weaned calves.

Diarrhea (scours) remains the predominant cause of mortality among dairycalves. There are multiple causes of scours including malabsorption andimproper nutrition; however, infections by bacteria, viruses, andprotozoa are the primary etiological agents. It is important to considerthat scours in calves may be due to a number of concurrentgastrointestinal insults by numerous pathogens. Susceptibility to acuteundifferentiated diarrhea can be largely determined by the quantity,quality, and administration time of colostrum.

The costs associated with scours are difficult to estimate; however,mortality alone represents a large expense, since, at birth, a heiferhas an estimated value of $400-$600. Scours does not always result indeath, but costs associated with treatment (e.g. electrolytes,antibiotics, veterinary services and associated labor) can besignificant. In addition, animal sickness and death can negativelyimpact the morale of farm laborers and must be taken into consideration,though the financial costs of this cannot be readily quantified.

Serum immunoglobulin obtained from colostrum can offer some limitedprotection to calves from bacterial and viral infections. However, thisprotective effect begins to diminish <96 hours after birth, which couldexplain the high onset of viral scours 5-7 days following birth.Prophylactic antibiotics and vaccines administered to calves arefrequent measures used to prevent scours in calves. While antibioticadministration can be effective against bacterial infections,antibiotics are ineffective against viruses and protozoa and, in fact,they can promote the development of viral or protozoal scours bydiminishing the normal protective flora. Vaccinations can also conferprotection against scours, however, the full protective immune responsedoes not occur until after few weeks of administration. Despite someadvances in prevention and treatment, the incidence of scours can varywildly between dairy herds.

A major factor contributing to the onset of scours in calves is thepractice of removing calves from their mother cows immediately afterbirth, and transporting them to facilities away from adult animals. Thegastrointestinal tracts of mammals, including calves, are sterile atbirth, but rapidly become colonized by microflora located near themother's vagina and anus. Other bacteria begin to establish themselveswhen the neonate comes into contact with new objects (feed, dirt, gates,fences, handlers, etc.). Prior to the current practice of removing acalf from its mother, protective microflora would become established inthe calf due to contact with the mother via licking, nursing, andgrooming. Thus, one possible avenue to reduce the incidence and severityof scours includes manipulating the microbial flora of a calves'digestive tract.

It has long been known that a number of beneficial bacteria colonize theintestinal tracts of mammals and can promote the well being of the host.It has also been recognized for many years that the consumption ofexogenous bacteria, often referred to as probiotics, can elicitbeneficial effects upon a host. In humans, these probiotic bacteria havebeen shown to reduce the severity and duration of rotaviral-induceddiarrhea, alleviate lactose intolerance, and enhance gastrointestinalimmune function. Traditionally, food sources such as yogurt have beenconsidered probiotic-carriers providing these health-promoting benefits.It is believed that the consumption of foods rich in probiotic bacteria,including lactic acid bacteria and bifidobacteria, leads to colonizationof the human gastrointestinal tract of humans.

It is also well established that the addition of probioticmicroorganisms to animal feed can improve animal efficiency and health.Specific examples include increased weight gain-to-feed intake ratio(feed efficiency), improved average daily weight gain, improved milkyield, and improved milk composition by dairy cows as described by U.S.Pat. Nos. 5,529,793 and 5,534,271. The administration of probioticorganisms can also reduce the incidence of pathogenic organisms incattle, as reported by U.S. Pat. No. 7,063,836.

Researchers have demonstrated that the consumption of probiotics byanimals used in food production can improve the efficiency of animalproduction. Probiotics may work by competitive exclusion in which livemicrobial cultures act antagonistically on specific organisms to cause adecrease in the numbers of that organism. Mechanisms of competitiveexclusion include production of antibacterial agents (bacteriocins) andmetabolites (organic acids and hydrogen peroxide), competition fornutrients, and competition for adhesion sites on the gut epithelialsurface. Lactic acid bacteria are generally considered as food gradeorganisms and there are many potential applications of protectivecultures in various foods. A number of different factors have beenidentified that contribute to the antimicrobial activity of lactic acidbacteria. These bacteria produce different antimicrobials, such aslactic acid, acetic acid, hydrogen peroxide, carbon dioxide andbacteriocins, which can inhibit pathogenic microorganisms.

Propionic acid is important in ruminal and intestinal fermentations andis a precursor to blood glucose synthesis. Several examples areavailable that demonstrate the positive impact of feeding propionicacid-producing organisms to cattle. For example, U.S. Pat. Nos.5,529,793 and 5,534,271, 6,455,063 and 6,887,489 demonstrate beneficialeffects of propionic acid-producing bacteria upon cattle growth. Lacticacid bacteria (LAB) can inhibit pathogens in various food sources.Lactic acid producing and lactate utilizing bacteria may also be helpfulin inhibiting pathogenic growth in animals and improving the productionof dairy products. Lactic acid producing and lactate utilizing bacteriaare beneficial for the utilization of feedstuffs by ruminants and havebeen fed to cattle to improve animal performance.

Propionic acid is important in ruminal and intestinal fermentations andis a precursor to blood glucose synthesis. Several examples areavailable that demonstrate the positive impact of feeding propionicacid-producing organisms to cattle. For example, U.S. Pat. Nos.5,529,793 and 5,534,271, issued to Garner and Ware, along with U.S. Pat.Nos. 6,455,063 and 6,887,489, issued to Rehberger et al., teach of thebeneficial effects that propionic acid-producing bacteria have uponcattle growth. Lactic acid bacteria (LAB) can inhibit pathogens invarious food sources. Lactic acid producing and lactate utilizingbacteria may also be helpful in inhibiting pathogenic growth in animalsand improving the production of dairy products. Lactic acid producingand lactate utilizing bacteria are beneficial for the utilization offeedstuffs by ruminants and have been fed to cattle to improve animalperformance.

Bacteriocins are ribosomally synthesized extracellularly releasedbioactive peptides or peptide complexes which have bacteriocidal orbacteriostatic activity. The producer cells exhibit immunity to theaction of its own bacteriocin. Bacteriocin producing strains can beidentified in a deferred antagonism assay where colonies of putativeproducer cells are covered with a bacterial strain which is sensitive tothe bacteriocins. After incubation, zones of inhibition are visible.Bacteriocins are known to inhibit foot borne pathogens such asClostridium botulinum, Enterococcus faecalis, Listeria monocytogenes andStaphylococcus aureus.

Four general classes of bacteriocins have been characterized: 1)lantibiotics, 2) small <13 kDa hydrophobic heat stable peptides, 3)large >30kDa heat labile proteins and 4) complex proteins that requireadditional carbohydrate or lipid moieties to attain antimicrobialactivity. Lantibiotics are a family of membrane active peptides thatcontain a thio-ether amino acid known as lanthionine and β-methyllanthionine as well as other modified amino acids such as dehydratedserine and threonine. A particular feature of lantibiotics is thepresence of post translationally modified amino acid residues. Oneexample of a lantibiotic is nisin. Bacteriocins which are small heatstable peptides do not contain modified amino acid residues. Large heatlabile bacteriocins include helviticin-J and lactacins A and B.

A majority of bacteriocins produced by bacteria are lantibiotics orsmall hydrophobic heat stable peptides. Nisin, a lantibiotic iseffective at inhibition of Gram-positive bacteria such as Bacillus andClostridium. However, Nisin has demonstrated no effectiveness againstGram-negative bacteria. Among the small hydrophobic heat stablepeptides, pediocins are frequently encountered and possess the abilityto inhibit Listeria monocytogenes.

Lactobacillus genus includes the most prevalently administered probioticbacteria (Flint and Angert 2005). Lactobacillus is a genus of more than25 species of gram-positive, catalase-negative, non-sporulating,rod-shaped organisms (Heilig et al., 2002). Lactobacillus speciesferment carbohydrates to form lactic acid as reported in U.S. Pat. No.7,323,166. Lactobacillus species are generally anaerobic, non-motile,and do not reduce nitrate as reported in U.S. Pat. No. 7,323,166.Lactobacillus species are often used in the manufacture of food productsincluding dairy products and other fermented foods as reported by Heiliget al., 2002 and U.S. Pat. No. 7,323,166. Lactobacillus species inhabitvarious locations including the gastrointestinal tracts of animals andintact and rotting plant material as reported by Heilig et al., 2002 andU.S. Pat. No. 7,323,166. Lactobacillus strains appear to be present inthe gastrointestinal tract of approximately 70% of humans that consume aWestern-like diet. Heilig et al., 2002. The number of Lactobacilluscells in neonates is approximately 10⁵ colony forming units (CFU) pergram CFU/g of feces. Heilig et al., 2002. The amount in infants of onemonth and older is higher, ranging from 10⁶ to 10⁸ CFU/g of feces.Heilig et al., 2002.

Lactic acid and products containing lactic acid have been found toenhance gains in the starting period of cattle (first 28 days) andreduce liver abscesses when given prior to the transition from aroughage diet to a feedlot diet. Various strains of Lactobacillusacidophilus have been isolated which restore and stabilize the internalmicrobial balance of animals. Manfredi et al., U.S. Pat. No. 4,980,164,is such a strain of Lactobacillus acidophilus which has been isolatedfor enhancing feed conversion efficiency. The Lactobacillus acidophilusstrain of the Manfredi et al patent, has been designated strain BT1386and received accession number ATCC No. 53545 from the American TypeCulture Collection in Rockville, Md. Strain ATCC 53545 demonstrates agreater propensity to adhere to the epithelial cells of some animalswhich would increase their ability to survive, initiate and maintain apopulation within an animal intestine. Thus, the primary mode of actionas previously understood relative to Lactobacillus acidophilus occurspost-ruminally.

The most common method used today to control pathogenic populations inlivestock is through the use of antimicrobial compounds. While these areeffective for short-term treatments, prolonged application ofantimicrobial compounds leads to the evolution of antibiotic resistancein pathogenic organisms. The widespread occurrence of antibioticresistant microorganisms is well known, some of the most common beingmethicillin resistant Staphylococcus aureus (MRSA) and vancomycinresistant enterococci (VRE). Bacteria are remarkably adaptable todeleterious environments with their abilities to rapidly reproduce andmodify their genetic content. Thus, it is inevitable that afterprolonged application of any method that disrupts or kills bacteria apopulation that is recalcitrant to its effects will eventually arise. Itis not uncommon now in the medical environment that doctors often resortto using multiple antibiotics concurrently or in succession to eradicatepathogenic organisms.

As with antibiotics, bacteria can also become resistant to otherbiological treatments. For example, bacteriophages are able to reducepathogen populations, but inevitably, a fraction of the targetedbacterial population is not infected. This small sub-population thenrapidly reproduces and attains sizable population numbers. Someresearchers have been able to overcome this by using multiple phages ina “cocktail” to reduce pathogen populations further than if only one wasused. The premise behind including multiple phage is that differentphage utilize different sites for attachment and infection of the hostbacterium. While a cell can become resistant to one phage by modifyingthe phage attachment site, it is more difficult for the bacterium tomodify two, three, or more attachment loci to evade all of the differentphage in the cocktail.

Similar circumstances have been seen with the application of probioticbacteria that are meant to inhibit or reduce the numbers of pathogenicbacteria within a gastrointestinal system. Some researchers havecommented that significantly better animal performance and pathogenreductions were seen in treated animals early in their experiments, butthe beneficial effects were no longer statistically different afterprolonged application of the probiotic product. It is possible that thetarget populations were initially affected, but prolonged usage of theprobiotic product led to the selection of bacterial populations thatwere not influenced by the application of the product. However, as seenwith multiple phage application, it may be possible to avoid theadaptation of pathogens to probiotic treatment with the inclusion ofmultiple strains of bacteria.

There are numerous advantages for the inclusion of multiple strains ofmicroorganisms in a microbial product. The potential advantagesdescribed below, whether working independently or concurrently, allowfor a superior microbial product and enhanced benefits for the host.

Different microbial strains utilize certain nutrients more efficientlythan others. The ability to use available nutrients in a gut environmentis necessary for the microbe to produce antimicrobial compounds or tobeneficially affect the host GI system. However, the nutrientavailability is constantly changing because of animal behavior,different foods consumed, antibiotic use, energy requirements, or healthof the animal. These fluctuations allow for different microbes toproliferate while other microbial populations diminish.

The use of different microbial strains also allows for the production ofdifferent microbial metabolites. Different metabolites have differenteffects upon pathogenic populations. Lactic acid is a powerfulantimicrobial agent against some pathogens, while propionic acid is moreeffective against other populations. It should also be considered thatjust as metabolites produced from cells from the microbial productaffect GI populations, endogenous microorganisms produce chemicals thatmay be inhibitory to some strains in the microbial product. Theinclusion of different strains in a product increases the likelihoodthat the product will have a positive effect.

Additionally, the production of bacteriocins is known to influencebacterial populations. There is a large diversity of bacteriocins knownand most target very specific microbial populations. Thus, a microbialproduct that contains multiple strains may be able to produce multiplebacteriocins and target different groups of pathogenic populations.Conversely, the intestinal tract contains a large diversity ofbacteriocin producing bacteria. While some of the produced bacteriocinsmay affect one of the included strains, it is unlikely to affect all ofthe included microorganisms.

Another benefit of a multiple-strain containing product is the abilityto target more than one pathogen population. Microbial pathogens arevery diverse are require different methods to reduce or eliminate theirpopulations. Thus, a product containing different microorganisms thatare able to effect different pathogenic populations will result in anoverall healthier system.

Different microorganisms positively influence the gastrointestinalsystem through different mechanisms. Including bacteria that workthrough different methods may result in a superior product. One strainmay reduce pathogen populations, while another has an immunostimulativeeffect, while another produces micronutrients essential for the host.Interestingly, multiple strains may also provide synergistic effectsupon the host or pathogen inhibition abilities. One strain alone may notbe able to reduce certain populations, but the combination of twodifferent strains working through different mechanisms can reducepathogen populations.

Additionally, the use of multiple beneficial microorganisms can helpovercome bacteriophages that infect and kill bacteria. Bacteriophagesare very common in gastrointestinal systems and have profound effectsupon the microbial community. Bacteriophages require specific sites on acell to bind and infect. Thus, including multiple microorganisms in aproduct, the greater the likelihood that at least some populations fromthe product will evade bacteriophage attack and elicit beneficialeffects upon the microbial community and host.

Some research has illustrated that a combination of different strains ofbeneficial probiotic bacteria can be used to treat discrete disorders.For example U.S. Pub. No. 20070280910 describes a probiotic compositionthat includes three different bacterial species consisting of Bacillussubtilis, Bacillus coagulans, and Enterococcus faecium purportedly totreat autism, yeast infections, fybromyalgia, and irritable bowelsyndrome. However, what is needed is a combination of probiotic bacteriacompositions for administration to animals to eliminate or reducegastrointestinal pathogens where a single probiotic bacteria species maybe resisted by evolving pathogenic bacteria or phages found in thegastrointestinal environment. The novel approach described here meetsthese needs by providing a microbial composition that will decrease theincidence of pathogens, decrease animal mortality, improve animalhealth, and maximize animal efficiency.

SUMMARY

Certain embodiments of the present disclosure concern a method ofinhibiting or reducing a population of pathogenic bacteria in or on ananimal. In such embodiments the population of pathogenic bacteria may beon the skin of the animal, in the blood of the animal or in one or moreorgans of the animal. In specific embodiments, the disclosure concerns amethod of inhibiting or reducing a population of pathogenic bacteria inthe gastrointestinal tract of the animal.

In embodiments concerning the inhibition or reduction of a population ofpathogenic bacteria, the method comprises providing to the animal two ormore administrations of a composition comprising at least one probioticmicroorganism.

In the embodiments of the disclosure, the probiotic microorganism may beany probiotic organism or any strain of a probiotic microorganism. Inpreferred embodiments, the composition to be administered comprisesprobiotic microorganisms which are lactic acid producing microorganisms.In specific embodiments the composition comprises Enterococcus faecium,Bacillus licheniformis, Lactococcus lactis, Bacillus subtilis,Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacteriumbifidum, Bifidobacterium infantis, Bifidobacterium longum,Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillusagilis, Lactobacillus alactosus, Lactobacillus alimentarius,Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillusamylovorus, Lactobacillus animalis, Lactobacillus batatas, Lactobacillusbavaricus, Lactobacillus bifermentans, Lactobacillus bifidus,Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus,Lactobacillus catenaforme, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus collinoides, Lactobacillus confusus,Lactobacillus coprophilus, Lactobacillus coryniformis, Lactobacilluscorynoides, Lactobacillus crispatus, Lactobacillus curvatus,Lactobacillus delbrueckii, Lactobacillus desidiosus, Lactobacillusdivergens, Lactobacillus enterii, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus frigidus, Lactobacillusfructivorans, Lactobacillus fructosus, Lactobacillus gasseri,Lactobacillus halotolerans, Lactobacillus helveticus, Lactobacillusheterohiochii, Lactobacillus hilgardii, Lactobacillus hordniae,Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillus jugurti,Lactobacillus kandleri, Lactobacillus kefir, Lactobacillus lactis,Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillusmalefermentans, Lactobacillus mali, Lactobacillus maltaromicus,Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis,Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum,Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillusrhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillustorquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillussalivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae,Lactobacillus sobrius, Lactobacillus trichodes, Lactobacillusvaccinostercus, Lactobacillus viridescens, Lactobacillus vitulinus,Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus zeae,Pediococcus acidlactici, Pediococcus pentosaceus, Streptococcuscremoris, Streptococcus diacetylactis, Streptococcus faecium,Streptococcus intermedius, Streptococcus lactis, Streptococcusthermophilus, Propionibacterium freudenreichii, Propionibacteriumacidipropionici, Propionibacterium jensenii, Propionibacterium thoenii,Propionibacterium cyclohexanicum, Propionibacterium granulosum,Propionibacterium microaerophilum, Propionibacterium propionicum,Propionibacterium acnes, Propionibacterium australiense,Propionibacterium avidum or a combination thereof. In other embodiments,the composition comprises one or more different strains of theaforementioned species of probiotic microorganisms.

In embodiments of the present disclosure wherein populations ofpathogenic bacteria are to be reduced or inhibited, the pathogenicbacteria may be any pathogenic bacteria known to afflict animals, suchas mammals. In particular embodiments the pathogenic bacteria isStaphylococcus aureus, Staphylococcus epidermis, Staphylococcussaprophyticus, Streptococcus pyogenes, Streptococcus pneumoniae,Streptococcus agalactiae, Enterococcus faecalis, Corynebacteriumdiptheriae, Bacillus anthracis, Listeria monocytogenes, Clostridiumperfringens, Clostridium tetanus, Clostridium botulinum, Clostridiumdifficile, Neisseria meningitidis, Neisseria gonorrhoeae, Escherichiacoli, Salmonella typhimurium, Salmonella cholerasuis, Salmonellaenterica, Salmonella enteriditis, Yersinia pestis, Yersiniapseudotuberculosis, Yersinia enterocolitica, Vibrio cholerae,Campylobacter jejuni, Campylobacter fetus, Helicobacter pylori,Pseudomonas aeruginosa, Pseudomonas mallei, Haemophilus influenzae,Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma urealyticum,Legionella pneumophila, Treponema pallidum, Leptospira interrogans,Borrelia burgdorferi, Mycobacterium tuberculosis, Mycobacterium leprae,Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae,Rickettsia ricketsii, Rickettsia akari, Rickettsia prowazekii, Brucellaabortus, Brucella melitens, Brucella suis, Francisella tularensis orcombinations thereof. Additionally, in certain embodiments, thepathogenic bacteria include one or more strains of one of theaforementioned pathogenic bacteria. For example, it is contemplated thatthe methods of the present disclosure may be used to reduce or inhibit apopulation of the O157:H7 strain of Escherichia coli.

In embodiments of the present disclosure wherein two or moreadministrations of a composition comprising at least one probioticmicroorganism is contemplated, specific embodiments contemplate thateach composition differs from the other or previously administeredcompositions by at least one species or strain of probioticmicroorganism.

In embodiments of the present disclosure wherein an administration ofprobiotic microorganisms is contemplated, the number of microorganismsper administration may be any amount capable of providing someinhibition or reduction of a population of pathogenic bacteria. Inspecific embodiments the number of microorganisms per administration isbetween 1×10³ and 1×10⁹ microorganisms. In preferably the number ofmicroorganisms in an administration is at approximately 1×10⁶microorganisms.

The administrations may be timed such that an administration isseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,17, 19, 20, 21, 22 or 23 hours or 1, 2, 3, 4, 5, or 6 days or 1, 2 or 3weeks or 1 month or some duration in between. In preferred embodimentsthe administrations are separated by 1 to 7 days. In specificembodiments, the administrations are separated by 1 day.

The administration may be oral, rectal, or via injection. In preferredembodiments, the administrations are oral administrations. Inembodiments wherein the administrations are oral administrations, thecomposition comprising one or more probiotic bacteria may be mixed withanimal feed or mixed with animal drinking water. In such embodiments,the composition or compositions may be formulated as a liquidformulation for administration, or as a freeze dried formulation, or asa gel formulation or as a spore formulation.

Additional steps in inhibiting or reducing population of pathogenicbacteria in an animal include assessing the presence of pathogenicbacteria in the gastrointestinal tract of the animal betweenadministrations. In more specific embodiments, the animal is assessedfor the presence of pathogenic bacteria, for strain of pathogenicbacteria, species of pathogenic bacteria and number of pathogenicbacteria present. In specific embodiments, this assessment is done byexamining the feces of the animal.

Consistent with long standing patent law, the words “a” and “an” denote“one or more,” when used in the text or claims of this specification inconjunction with the word “comprising” or where the context of the usagesuggests that, from either a grammatical or scientific standpoint, thesewords should so denote.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating the growth inhibition of E. colistrain CKYL1 by various lactic acid producing bacteria.

FIG. 2 is a bar graph illustrating the growth inhibition of E. colistrain 25922 by various lactic acid producing bacteria.

FIG. 3 is a bar graph illustrating the growth inhibition of E. colistrain 0157:H7 by various lactic acid producing bacteria.

FIG. 4 is a bar graph illustrating the growth inhibition of Salmonellacholerasuis by various lactic acid producing bacteria.

FIG. 5 is a bar graph illustrating the growth inhibition of Salmonellatyphimurium by various lactic acid producing bacteria.

DETAILED DESCRIPTION

a. Definitions

Before describing the present disclosure in detail, it is to beunderstood that this disclosure is not limited to particularcompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Also, throughout this specification, variouspublications are referenced. The disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich the disclosed subject matter pertains. The references disclosedare also individually and specifically incorporated by reference hereinfor the material contained in them that is discussed in the sentence inwhich the reference is relied upon.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are now described.

In this specification and the claims that follow, reference will be madeto a number of terms which may be considered to have the followingmeanings:

By “reduce” or other forms of the word, such as “reducing” or“reduction,” may in certain instances refer to lowering of an event orcharacteristic (e.g., microorganism growth or survival). It isunderstood that this is typically in relation to some standard orexpected value, in other words it is relative, but that it is not alwaysnecessary for the standard or relative value to be referred to. Forexample, “reduces the population of bacteria” in certain instances mayrefer to lowering the amount of bacteria relative to a standard or acontrol.

By “treat” or other forms of the word, such as “treated” or “treatment,”may, in certain instances mean to administer a composition or to performa method in order to reduce, prevent, inhibit, break-down, or eliminatea particular characteristic or event (e.g., microorganism growth orsurvival).

The term “viable cell” may in certain instances mean a microorganismthat is alive and capable of regeneration and/or propagation, while in avegetative, frozen, preserved, or reconstituted state.

The term “viable cell yield” or “viable cell concentration” may, incertain instances refer to the number of viable cells in a liquidculture, concentrated, or preserved state per a unit of measure, such asliter, milliliter, kilogram, gram or milligram.

The term “cell preservation” in certain instances may refer to a processthat takes a vegetative cell and preserves it in a metabolically inertstate that retains viability over time. As used herein, the term“product” in certain instances may refer to a microbial composition thatcan be blended with other components and contains specifiedconcentration of viable cells that can be sold and used.

As used herein, the terms “microorganism” or “microbe” may in certaininstances refer to an organism of microscopic size, to a single-celledorganism, and/or to any virus particle. Our definition of microorganismincludes Bacteria, Archaea, single-celled Eukaryotes (protozoa, fungi,and ciliates), and viral agents. The term “microbial” is used herein todescribe processes or compositions of microorganisms, thus a“microbial-based product” is a composition that includes microorganisms,cellular components of the microorganisms, and/or metabolites producedby the microorganisms.

Microorganisms can exist in various states and occur in vegetative,dormant, or spore states. Microorganisms can also occur as either motileor non-motile, and may be found as planktonic cells (unattached),substrate affixed cells, cells within colonies, or cells within abiofilm.

The term “prebiotic” in certain instances may refer to food ingredientsthat are not readily digestible by endogenous host enzymes and conferbeneficial effects on an organism that consumes them by selectivelystimulating the growth and/or activity of a limited range of beneficialmicroorganisms that are associated with the intestinal tract.

The term “probiotic” in certain instances may refer to one or more livemicroorganisms that confer beneficial effects on a host organism.Benefits derived from the establishment of probiotic microorganismswithin the digestive tract include reduction of pathogen load, improvedmicrobial fermentation patterns, improved nutrient absorption, improvedimmune function, aided digestion and relief of symptoms of irritablebowel disease and colitis.

The term “synbiotic” in certain instances may refer to a compositionthat contains both probiotics and prebiotics. Synbiotic compositions arethose in which the prebiotic compound selectively favors the probioticmicroorganism.

The term “gastrointestinal tract” in certain instances may refer to thecomplete system of organs and regions that are involved with ingestion,digestion, and excretion of food and liquids. This system generallyconsists of, but not limited to, the mouth, esophagus, stomach and orrumen, intestines (both small and large), cecum (plural ceca),fermentation sacs, and the anus.

The term “pathogen” in certain instances may refer to any microorganismthat produces a harmful effect and/or disease state in a human or animalhost.

The term “fermentation” in certain instances may refer to a metabolicprocess performed by an organism that converts one substrate to anotherin which the cell is able to obtain cellular energy, such as when anorganism utilizes glucose and converts it to lactic acid or propionicacid. Many of the end-substrates formed in fermentation processes arevolatile fatty acids.

The term “volatile fatty acids” in certain instances may refer toshort-chain fatty acids containing six or fewer carbon atoms and atleast one carboxyl group. Some examples of VFAs include, but are notlimited to: lactic acid, acetic acid, propionic acid, butyric acid,isobutyric acid, valeric acid, and isovaleric acid, which are productsof microbial fermentation within the digestive tracts of animals.Volatile fatty acids can be absorbed through the intestines of animalsand used as an energy or carbon source. Microbes produce VFAs based onavailable substrates and also rely upon VFAs for energy and carbonsources.

The term “lactic acid” in certain instances may refer to a byproduct ofglucose fermentation resulting in a three-carbon acid with the chemicalformula C₃H₆O₃. This includes, but is not limited to, lactic acidderived from specific strains of bacteria or lactic acid derived fromother types of organisms. Lactic acid can be microbialstatic,microbialcidal, bacteriostatic, bacteriocidal or bacteriolytic; theseconcepts are known to skilled persons. “Lactic acid producing” refers toany organism that generates lactic acid.

The term “bacteriocin(s)” in certain instances may refer to (poly)peptides and proteins that inhibit one or more microbial species. Thisincludes, but is not limited to, (poly) peptides or proteins that werederived from specific strains of bacteria or (poly) peptides that arederived from other types of organisms. The bacteriocin can bemicrobialstatic, microbialcidal, bacteriostatic, bacteriocidal, orbacteriolytic; these concepts are known to skilled persons. For thetreatment of produce and other food products the bacteriocin ispreferably microbialcidal or bacteriocidal. “Bacteriocin producing” incertain instances may refer to any organism that generates bacteriocins.

As used herein, “hydrogen peroxide” in certain instances may refer to abyproduct of oxygen metabolism that has the chemical formula H₂O₂. Thisincludes, but is not limited to, hydrogen peroxide derived from specificstrains of bacteria or hydrogen peroxide derived from other types oforganisms. Hydrogen peroxide can be microbialstatic, microbialcidal,bacteriostatic, bacteriocidal or bacteriolytic; these concepts are knownto skilled persons. “Hydrogen peroxide-producing” refers to any organismthat generates hydrogen peroxide.

As used herein, the term “synergistic” in certain instances may refer toa property wherein the combined result of two effects is greater thanwould be expected if the two effects were added together. The term“synergistically” is used to describe a synergistic effect.

As used herein, the phrase “foregut fermentor” in certain instances mayrefer to an animal having an anatomical compartment in the alimentarycanal that is positioned anterior to the stomach that is used formicrobial fermentation and digestion of ingested materials. Ruminalfermentors are considered foregut-fermenting organisms.

As used herein, the phase “ruminal fermentor” or “rumen fermenting” incertain instances may refer to an animal having a large,multi-compartmented section of the digestive tract, called a rumen,which is positioned between the esophagus and the anus. Rumen are verycomplex ecosystems that support microbial fermentation of cellulose,plant matter, and other ingested materials. Ruminal-fermentors may alsobe termed “cranial fermentors” or “ruminants”. Some examples ofrumen-fermenting organisms include cattle, sheep, goats, camels, llama,bison, buffalo, deer, wildebeest, antelope, etc.

As used herein, the phrase “hindgut fermentor” in certain instances mayrefer to an animal having a complex large intestine that may or may notinclude specialized fermentation chambers that can include a cecum orblind sac, that is positioned posterior to the stomach in the alimentarycanal. Cecal fermentors and intestinal fermentors are both consideredhindgut-fermenting organisms.

As used herein, the phrase “cecal fermentor” in certain instances mayrefer to an animal having a complex large intestine that includes acecum or a blind sac along the digestive tract. The cecum of a cecalfermentor forms a distinct chamber, which is the primary site ofmicrobial fermentation of cellulose, plant matter, or other ingesta. Acecal-fermentor may also be referred to as “caudal fermentor”.Cecal-fermentors include horses, elephants, rabbits, mice, rats, guineapigs, etc.

As used herein, the term “intestinal fermentor” in certain instances mayrefer to an animal that does not primarily rely upon microbialfermentation of ingesta in a rumen or large cecum. In the digestivetracts of intestinal fermentors, microbial fermentation occurs primarilywithin the large intestine or colon. Intestinal fermentors includechickens, pigs, humans, etc.

As used herein, the term “monogastric” in certain instances may refer toan animal having a single, simple (single chambered) stomach. Typically,cecal fermentors and intestinal-fermentors are monogastric animals. Someexamples of monogastric animals include horses, chickens, pigs, humans,etc.

As used herein, the term “polygastric” in certain instances may refer toan animal having a multiple, complex (multi-chambered) stomachs. Ruminalfermentors are polygastric animals.

As used herein, the phrase “pre-gastric fermentation” in certaininstances may refer to microbial fermentation that occurs before thefood reaches a ‘true’ stomach, which is generally the site of gastricacid and digestive enzyme secretion. Ruminants are pre-gastricfermentors.

As used herein, the phrase “post-gastric fermentation” in certaininstances may refer to microbial fermentation that occurs after foodpasses through a stomach, which is generally the site of gastric acidand digestive enzyme secretion. Hindgut fermentors, including cecalfermentors and intestinal fermentors, utilize post-gastric fermentation.

As used herein, the term “herbivore” in certain instances may refer toan animal that exclusively consumes plant material.

As used herein, the term “omnivore” in certain instances may refer to ananimal that consumes both plant and animal material.

As used herein, the term “carnivore” in certain instances may refer toan animal that exclusively consumes animal material.

As used herein, “digesta” in certain instances may refer to food or anyother material that enters the alimentary canal and undergoes,completely or partially, through the process of being digested or brokendown into smaller components.

b. Introduction

The present disclosure discloses a novel synbiotic composition comprisedof multiple probiotic microorganisms. The present disclosure can beadjusted to provide beneficial effects to many types of animals,including ruminal fermentors, cecal fermentor and intestinal fermentors.In one preferred embodiment, the probiotic formulation supplemented withprebiotic compounds is fed to ruminal fermentors to reduce scours eventsand improve animal health. Ruminal fermentors that might benefit fromthe present disclosure include but are not limited to: cattle, sheep,goats, camels, llama, bison, buffalo, deer, wildebeest, antelope, andany other pre-gastric fermentor. In another embodiment, the probioticformulation supplemented with prebiotic compounds is fed to cecalfermentors to reduce scours events and improve animal health. Cecalfermentors that might benefit from said disclosure include but are notlimited to: horses, ponies, elephants, rabbits, hamsters, rats, hyraxes,guinea pigs, and any other post-gastric fermentor that using the cecumas the primary location of fermentative digestion. In anotherembodiment, the probiotic formulation supplemented with prebioticcompounds is fed to intestinal fermentors to reduce scours events andimprove animal health. Intestinal fermentors that might benefit fromsaid disclosure include but are not limited to: humans, pigs, chickens,and other post-gastric fermentor using the large intestine as theprimary location of fermentative digestion. In each case, the synbioticcomposition is packaged in format that ensures survival of both theprobiotic and prebiotic components into the gastrointestinal system ofthe animal.

One object of the present disclosure to provide a novel composition thatwill reduce mortality and morbidity and/or improve animal health and/orproductivity. A further object of the present disclosure to provide acomposition that will be used once, periodically or on a continual basisto reduce the incidence and severity of scours and/or improve animalhealth. Still further, another object of the present disclosure is toprovide a composition that is durable and easy to apply to animal feedor other easily ingestible materials.

The novel composition, which comprises mixtures of probioticmicroorganisms, facilitates the production of lactic acid, whichinhibits the growth of pathogenic organisms during digestivefermentation. The composition comprises a mixture of lacticacid-producing bacteria in combination with a lactic acid-utilizing andpropionic acid-producing bacteria. In some embodiments, the compositionis mixed with colostrum or milk replacers. In other embodiments themixture is applied to milk or water administered to the calves. Animalscan be treated with one or more viable microorganisms in combinationwith prebiotic compounds to improve animal efficiency and/or health.Additive, or more preferably super-additive or more preferablysynergistic effects can be achieved with the administration of one ormore microbial species and/or strains. Animals can be treated once,multiple times, or continuously in a day.

It is preferred that the probiotic mixture could contain any number ofmicroorganisms and or microbial components and/or metabolites. Examplesof bacterial species that could be used for the probiotic mixtureinclude but are not limited to the group consisting of: Enterococcusfaecium, Bacillus licheniformis, Lactococcus lactis, Bacillus subtilis,Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacteriumbifidum, Bifidobacterium infantis, Bifidobacterium longum,Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillusagilis, Lactobacillus alactosus, Lactobacillus alimentarius,Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillusamylovorus, Lactobacillus animalis, Lactobacillus batatas, Lactobacillusbavaricus, Lactobacillus bifermentans, Lactobacillus bifidus,Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus,Lactobacillus catenaforme, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus collinoides, Lactobacillus confusus,Lactobacillus coprophilus, Lactobacillus coryniformis, Lactobacilluscorynoides, Lactobacillus crispatus, Lactobacillus curvatus,Lactobacillus delbrueckii, Lactobacillus desidiosus, Lactobacillusdivergens, Lactobacillus enterii, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus frigidus, Lactobacillusfructivorans, Lactobacillus fructosus, Lactobacillus gasseri,Lactobacillus halotolerans, Lactobacillus helveticus, Lactobacillusheterohiochii, Lactobacillus hilgardii, Lactobacillus hordniae,Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillus jugurti,Lactobacillus kandleri, Lactobacillus kefir, Lactobacillus lactis,Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillusmalefermentans, Lactobacillus mali, Lactobacillus maltaromicus,Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis,Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum,Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillusrhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillustorquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillussalivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae,Lactobacillus sobrius, Lactobacillus trichodes, Lactobacillusvaccinostercus, Lactobacillus viridescens, Lactobacillus vitulinus,Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus zeae,Pediococcus acidlactici, Pediococcus pentosaceus, Streptococcuscremoris, Streptococcus discetylactis, Streptococcus faecium,Streptococcus intermedius, Streptococcus lactis, Streptococcusthermophilus, Propionibacterium spp., Propionibacterium freudenreichii,Propionibacterium acidipropionici, Propionibacterium jensenii,Propionibacterium thoenii, Propionibacterium cyclohexanicum,Propionibacterium granulosum, Propionibacterium microaerophilum,Propionibacterium propionicum, Propionibacterium acnes,Propionibacterium australiense, Propionibacterium avidum and strainsand/or combinations thereof. Furthermore, a lactic acid producingmicroorganism can be a strain of Lactobacillus spp., such as the MRL1,M35, LA45, LA51, L411, NPC 747, NPC 750, D3, and L7 strains. Examples ofa lactic acid-utilizing and/or propionic acid-producing organism includethe Propionibacterium spp. strains PF24, P5, P63, P1, and MRP1.

c. Formulations

In certain aspects of the disclosure contemplate a carrier formulationfor the probiotic microorganisms. In certain aspects of the disclosure,the carrier may be any number of different percentages (weight perweight, weight per volume, or volume per volume) of the final product.The carrier can comprise any amount of about 99.9%, about 95%, about90%, about 80%, about 70%, about 60% about 50%, about 40%, about 30% andso on. The remaining composition can also include other carriers such aslactose, glucose, sucrose, salt, cellulose, etc. In specific aspects ofthe disclosure, the carrier may be 50% or more of the total product.

In certain aspects of the disclosure, the carrier and composition canalso have defined properties, such as solubility/insolubility in wateror solubility/insolubility in fat, etc.

In certain aspects of the disclosure, other chemicals or materialsprincipally used for the reduction or absorption of moisture may also beincluded. These may include, but are not limited to: calcium stearate,sodium aluminosilicate, silica, calcium carbonate, zeolite,bicarbonates, sodium sulfate, silicon dioxide, or ascorbic acid.

In certain aspects of the disclosure, other chemicals or materialsprincipally used for the reduction or absorption of oxygen may also beincluded. These may include, but are not limited to, iron oxides,ascorbic acid, sodium sulfide, and silica materials.

In certain aspects of the disclosure, the biological material mixed withthe present carrier can be stored in a pouch or bag fabricated fromvarious materials, a bottle fabricated from a variety of materials, acapsule, a box, or other storage container.

In certain aspects of the disclosure, the biological material mixed inthe present carrier may also be used for the application onto a varietyof foods including, but not limited to, meats, vegetables, fruits,processed foods, or others.

In certain aspects of the present disclosure wherein formulations arecontemplated for preservation, such preservation may include a processof freezing, freeze-drying and/or spray-drying. In certain aspects, thepreserved bacteria contain a viable cell concentration of 1×10⁸ to5×10¹² cfu/g. Still further, in certain aspects the concentrations rangefrom 5×10¹⁰ cfu/g to 5×10¹³ cfu/g of bacteria.

In certain instances, a bacterial formulation for administration to asubject or a surface or other target can include a preservation matrix,which contains and preserves the bacterial culture. Such a matrix mayinclude a biologically active binding agent, an antioxidant, a polyol, acarbohydrate and a proteinaceous material. For example, the matrix mayhave a pH of from about 5.0 to about 7.0. Such a preservation matrix maybe capable of maintaining at least about 10⁶ viable cells for a periodof at least about 12 months in vitro. In other examples, such a matrixmaintains at least about 10⁷ viable cells for a period of at least about12 months in vitro, and more preferably, at least about 10⁸ viable cellsfor a period of at least about 12 months in vitro. A preservation matrixmay be comprised of ingredients to minimize the damaging effectsencountered during the preservation process and to provide functionalproperties. For example when a Lactobacillus strain of the presentdisclosure is added to a preservation matrix for preservation, it is mayconverted from an actively growing metabolic state to a metabolicallyinactive state. In formulations of the present disclosure wherein apreservation matrix is contemplated, a biologically acceptable bindingagent can be used to both affix the bacterial culture or cultures to aninert carrier during a preservative process and to provide protectiveeffects (i.e., maintains cell viability) throughout preservation andstorage of the microbial cells. Preferred biologically acceptablebinding agents for use in a preservation matrix include, but are notlimited to a water-soluble gum, carboxymethyl cellulose and/or gelatin.A biologically acceptable binding agent typically comprises from about10% to about 20% by weight of the preservation matrix, and preferablycomprises about 14% by weight of the preservation matrix. In oneembodiment, a preservation matrix of the present disclosure comprisesabout 14% gelatin by weight of the preservation matrix.

Antioxidants included in a preservation matrix may be provided to retardoxidative damage to the microbial cells during the preservation andstorage process. A particularly preferred antioxidant is sodiumascorbate. An antioxidant typically comprises from about 0.1% to about1.0% by weight of the preservation matrix, and preferably comprisesabout 0.5% by weight of the preservation matrix. In one embodiment, apreservation matrix of the present disclosure comprises about 0.5%sodium ascorbate by weight of the preservation matrix.

Polyols (i.e., polyhydric alcohols) included in a preservation matrixmay be provided to maintain the native, uncollapsed state of cellularproteins and membranes during the preservation and storage process. Inparticular, polyols interact with the cell membrane and provide supportduring the dehydration portion of the preservation process. Preferredpolyols include, but are not limited to xylitol, adonitol, glycerol,dulcitol, inositol, mannitol, sorbitol and/or arabitol. A polyoltypically comprises from about 1% to about 25% by weight of thepreservation matrix, and preferably comprises about 6% by weight of thepreservation matrix. In one embodiment, a preservation matrix of thepresent disclosure comprises about 6% xylitol by weight of thepreservation matrix.

Carbohydrates included in a preservation matrix may be provided tomaintain the native, uncollapsed state of cellular proteins andmembranes during the preservation and storage process. In particular,carbohydrates provide cell wall integrity during the dehydration portionof the preservation process. Preferred carbohydrates include, but arenot limited to dextrose, lactose, maltose, sucrose, fructose and/or anyother monosaccharide, disaccharide or polysaccharide. A carbohydratetypically comprises from about 0.5% to about 5% by weight of thepreservation matrix, and preferably comprises about 2.5% by weight ofthe preservation matrix. In one embodiment, a preservation matrix of thepresent disclosure comprises about 2.5% dextrose by weight of thepreservation matrix.

A proteinaceous material included in a preservation matrix may providefurther protection of the microbial cell during the dehydration portionof the preservation process. Preferred proteinaceous materials include,but are not limited to skim milk and albumin. A proteinaceous materialtypically comprises from about 0.5% to about 5% by weight of thepreservation matrix, and preferably comprises about 1.5% by weight ofthe preservation matrix. In one embodiment, a preservation matrix of thepresent disclosure comprises about 1.5% skim milk by weight of thepreservation matrix.

One example of a method of preserving microbial cells within apreservation matrix includes coating the cell matrix suspension onto aninert carrier that preferably is a maltodextrin bead. The coated beadscan then be dried, preferably by a fluid bed drying method. Fluid beddrying methods are well known in the art. For example, maltodextrinbeads may be placed into a fluid bed dryer and dried at 33° C. The airpressure may be set to 1 bar, the cell suspension matrix can then besprayed onto the beads and the heat is increased to 38° C. The coatedbeads are then allowed to dry for an additional period of time. Thecoated maltodextrin beads can be stored as a powder, placed into gelatincapsules, or pressed into tablets.

In other formulations of the disclosure, the single strains orcombinations of strains of bacteria contemplated to be cultured can beformulated as a hard gelatin capsule. Gelatin capsules are commerciallyavailable and are well known in the art. In this embodiment, the abovepreservation method further comprises dispensing the cell suspensionmatrix to a gelatin capsule, chilling the gelatin capsule until the cellsuspension matrix forms a non-fluid matrix and to affix the gel to theinterior wall of the gelatin capsule, and desiccating the gelatincapsule in a desiccation chamber. The step of dispensing can beaccomplished by any means known in the art, and includes manual,semi-automated and automated mechanisms. The chilling step is performedat from about 4° C. to about 6° C. The step of desiccating the gelatincapsule can include the steps of (i) providing dry air to thedesiccation chamber containing less than about 25% moisture, at atemperature from about 24° C. to about 32° C.; and (ii) removinghumidified air from the desiccation chamber.

In this formulation of the present disclosure the desiccation processmay proceed for about 1 to about 6 hours. The desiccation chamber caninclude a compressor, at least one hydrocarbon scrubbing filter and achilled air compressor with or without a desiccant silica gel (or anyother suitable desiccant material) column, in series. The air enteringthe chamber (dry air) preferably contains less than about 25% moisture,and more preferably less than about 15% moisture, and even morepreferably less than about 5% moisture, down to as little as zeromoisture. The dry air should preferably have a temperature from about24° C. to about 32° C. This method allows preservation of microbialcells in a controlled environment with room temperature air in a shortperiod of time. Further examples of embodiments of preservation matricesand gelatin capsule formulations may be found in U.S. Pat. No. 6,468,526which is herein incorporated by reference in its entirety.

In certain applications, the bacteria cultured with the methodsdescribed herein may be placed in a microencapsulation formulation. Suchmicroencapsulation formulations may have applicability for example inadministration to subjects via oral, nasal, rectal, vaginal or urethralroutes. Spray drying is the most commonly used microencapsulation methodin the food industry, is economical and flexible, and produces a goodquality product. The process involves the dispersion of the corematerial into a polymer solution, forming an emulsion or dispersion,followed by homogenisation of the liquid, then atomisation of themixture into the drying chamber. This leads to evaporation of thesolvent (water) and hence the formation of matrix type microcapsules.

For example O'Riordan et al., 2001 reported microencapsulation and spraydrying of Bifidobacterium cells with a spray inlet temperature of 100°C. and low outlet temperature of 45° C. The cells were reported to beencapsulated satisfactorily to produce micro spheres with gelatinizedmodified starch as a coating material (O'Riordan et al., 2001). In thisstudy, spray drying was found to be a valuable process for encapsulatingBifidobacteria. The process of spray drying is economical, easily scaledup and uses equipment readily available in the food industry (Gibbs etal., 1999). A previous report indicated that survival of probioticbacteria during spray drying decreased with increasing inlettemperatures (Mauriello et al., 1999).

In one such example of microencapsulation, lyophilized bacteria aresuspended in 10 ml of 5% glucose saline solution in a volume so as toobtain a heavy suspension of bacteria which contains approximately 10⁹organisms per ml, at 0° C. to 4° C. The suspension of bacteria may thenbe rapidly, but gently, stirred while 0.2-0.4 ml of sodium alginatesolution (1.5% weight by volume) is added. The above mixture may then betransferred into a sterile container by using a nitrogen stream througha 14 gauge sheathed needle. The mixture may then be forced through a 30gauge multi-beveled needle under pressure using a large syringe andnitrogen stream. Very small droplets are generated at the end of theneedle, which are then dried by the nitrogen and air stream around the30 gauge needle, and the droplets are collected in an aqueous solutionof 1.3-2% calcium chloride where they gel. Thereafter, they are washedat least three times with 0.08-0.13% 2-(N-cyclohexyl-amino)ethanesulfonic acid (CHES) solution and 1.0-1.5% calcium chloridesolution. The gelled droplets or little spheres are further washed withat least a five-fold excess of the 0.1% CHES 1.1% calcium chloride, andnormal saline solution. The resultant spheres are then “snap frozen” inliquid nitrogen and then lyophilized. After these steps, theencapsulated organisms can be used in the formulations of the presentdisclosure. Other examples of microencapsulation can be found forexample in U.S. Pat. No. 5,641,209 that is herein incorporated byreference.

Dry microorganism cultures may be prepared according to the disclosure,in addition to any constituents present from a fermentation medium, suchas metabolic products, the medium may comprise at least one matrixmaterial with or without other stabilizing substances. These materialsare preferably selected from inorganic salts or buffers, at least oneother compound which is selected from mono-, oligo- and polysaccharides,polyols, polyethers, amino acids, oligo- and polypeptides, milk-derivedcompounds, organic carboxylic acids, mineral compounds, organic carriermaterials such as wheat semolina bran, alginates, DMSO, PVP(polyvinylpyrrolidone), CMC (carboxymethylcellulose), alpha-tocopherol,beta.-carotene and mixtures thereof.

Examples of suitable saccharide carrier components are sucrose,fructose, maltose, dextrose, lactose and maltodextrin. An example of asuitable polyol is glycerol. Examples of suitable amino acids areglutamic acid, aspartic acid and the salts thereof. An example of asuitable peptide carrier is peptone. An example of a milk-derivedcompound is, in addition to the abovementioned maltodextrin, also sweetwhey powder. Suitable organic carboxylic acids are, for example, citricacid, malic acid and L-ascorbic acid. Examples of suitable mineralcarriers are montmorillonite and palygorskite.

In certain aspects of the disclosure mixtures of the abovementionedclasses of substances may be employed. Mixtures of this type preferablycomprise, as main component, a matrix material, such as one of theabovementioned saccharide components or, for example, sweet whey powder,with or without a minor content of at least one further component, suchas a buffer component (for example citric acid) or an antioxidant (forexample L-ascorbic acid or α-tocopherol). The addition of furtherstabilizing constituents, such as sodium glutamate and/or peptone, haslikewise proved to be advantageous.

The matrix component is customarily used in carrier compositions usableaccording to the disclosure in about 5 to 30 times the amount of theother carrier constituents. Examples of particularly suitable carriercombinations are: a) sweet whey powder/citric acid/L-ascorbic acid(weight ratio about 40:1:1). b) maltodextrin/lactose/citricacid/L-ascorbic acid (weight ratio about 20:20:1:1), unsupplemented orsupplemented by about 1.5 parts of beta-carotene and 0.5 part ofalpha-tocopherol per part of citric acid. c) maltodextrin/sodiumglutamate/L-ascorbic acid (weight ratio about 10:1.5:1). d)lactose/glucose/peptone/citric acid (weight ratio about 6:6:1.2:1).

The carrier substances according to the disclosure can be added to themicroorganism suspension either as solid or in dissolved form. However,preferably, a sterile solution of the carrier/carriers is prepared, thisis cooled to a temperature of from 4 to 10° C. and this is mixed withthe likewise cooled microorganism suspension with gentle stirring. Toprepare a homogeneous suspension, the resultant mixture is stirred withfurther cooling for a period of from about 10 minutes to 1 hour.

The microorganism suspension containing the carrier added in the mannerdescribed above can then be dried in various ways. Suitable dryingprocesses are in principle freeze drying, fluidized-bed drying and,preferably, spray-drying. For the purposes of the present disclosure,spray-drying also comprises modified spray-drying processes, such asspray-agglomeration or agglomerating spray-drying. The latter process isalso known under the name FSD (fluidized spray-dryer) process.

Freeze-drying for preparing dry microorganism cultures according to thedisclosure can be carried out, for example, on the basis of thefreeze-drying process described in U.S. Pat. No. 3,897,307. The contentsof these publications are hereby incorporated completely by reference.

Another, drying process contemplated for use in the present disclosureis spray-drying. Those methods which can be used according to thedisclosure are essentially all spray-drying techniques known in the art.The material to be sprayed can, for example, be dried concurrently orcountercurrently; spraying can be carried out by means of asingle-component or multiple-component nozzle or by means of an atomizerwheel.

Preference is given according to the disclosure to the use of materialto be sprayed having a solids content (after addition of the carrier) offrom about 10 to 40, such as from about 10 to 25% by weight.

One particular factor according to the disclosure is the use ofpreconditioned, i.e. low-moisture, drying air. Preferably, use is madeof compressed air having a dew point at about −25° C.

The drying process according to the disclosure may be carried out insuch a manner that a very low residual moisture content is present inthe dry material. The percentage water content is preferably from about2 to 3% by weight. This may be achieved by adding a post-drying stepsubsequently to the spray-drying step. The drying material for thispurpose is, for example, post-dried in a fluidized bed, preferably at atemperature in the range of from 15 to 50° C. for a period of, forexample, from 15 minutes to 20 hours. Again, preferably, conditionedcompressed air or conditioned nitrogen serves as drying gas. However,the post-drying can also be performed by applying a vacuum of from about1 to 50 mm Hg for a period of from about 15 minutes to 20 hours and at atemperature of from about 15 to 50° C. In this case, preference is givento stirring the drying material, for example, using a paddle agitator.

Instead of the above-described physical post-drying processes, it isalso conceivable to add specific desiccants to the dry material obtainedfrom the spray-drying. Examples of suitable desiccants are inorganicsalts, such as calcium chloride and sodium carbonate, organic polymers,such as the product obtainable under the trade name Kollidion 90 F, andsilicon-dioxide-containing desiccants, such as silica gel, zeolites anddesiccants which are obtainable under the trade name Tixosil 38,Sipernat 22 S or Aerosil 200.

The content of viable microorganisms is in the range of from about 5×10⁵to 1×10¹² cfu/g of dry matter. These preparations are also calledaccording to the disclosure powder concentrates. Since, for individualfinal applications, lower contents of viable microorganisms are alsocompletely sufficient, powder concentrates of this type can therefore ifappropriate be blended to the final count of viable microorganisms bymixing with further inert carrier material.

Some bacteria can survive environmental stresses through the formationof spores. This complex developmental process is often initiated inresponse to nutrient deprivation. It allows the bacterium to produce adormant and highly resistant cell. Spores can survive environmentalassaults that would normally kill other bacteria. Some stresses thatendospores can withstand include exposure to high temperatures, high UVirradiation, desiccation, chemical damage and enzymatic destruction. Theextraordinary resistance properties of endospores make them ofparticular importance because they are not readily killed by manyantimicrobial treatments. Common bacteria that form spores includespecies from the Bacillus and Clostridium genera. Spores formed by thesebacteria remain in their dormant state until the spores are exposed toconditions favorable for growth. The inclusion of spores in a probioticcomposition is appealing because of their ability to withstandprocessing methods and can have extended shelf life viabilities.Additionally, bacterial spores can require less processing because theydo not require additional steps for preservation (such as freeze drying,spray drying, freezing, etc.) as is required for many other probioticorganisms.

In still other embodiments of the disclosure, a gel formulation fordelivery of probiotic bacteria to animals is used. A gel is definedherein as an apparently solid, jelly-like material formed from acolloidal solution. A colloidal solution is a solution in which finelydivided particles which are dispersed within a continuous medium in amanner that prevents them from being filtered easily or settled rapidly.Methods pertaining to the formulation of gels are set forth in U.S. Pat.No. 6,828,308, U.S. Pat. No. 6,280,752, U.S. Pat. No. 6,258,830, U.S.Pat. No. 5,914,334, U.S. Pat. No. 5,888,493, and U.S. Pat. No.5,571,314, each of which is herein specifically incorporated byreference in its entirety.

d. Uses of Formulated Bacterial Products

The methods and formulations of the present disclosure can be adjustedto provide beneficial effects to many types of animals, includingruminal fermentors, cecal fermentor and intestinal fermentors. In onepreferred embodiment, the product is fed to ruminal fermentors to reducescours events, improve animal health and animal productivity. Ruminalfermentors that might benefit from the present disclosure include butare not limited to: cattle, sheep, goats, camels, llama, bison, buffalo,deer, wildebeest, antelope, and any other pre-gastric fermentor. Inanother embodiment, the product is fed to cecal fermentors to reducescours events, improve animal health and animal productivity. Cecalfermentors that might benefit from the present disclosure include butare not limited to: horses, ponies, elephants, rabbits, hamsters, rats,hyraxes, guinea pigs, and any other post-gastric fermentor that usingthe cecum as the primary location of fermentative digestion. In anotherembodiment, product is fed to intestinal fermentors to reduce scoursevents, improve animal health and animal productivity. Intestinalfermentors that might benefit from said disclosure include but are notlimited to: humans, pigs, chickens, and other post-gastric fermentorusing the large intestine as the primary location of fermentativedigestion.

The various embodiments of the disclosure include the application of acombination of probiotic microorganisms to the animal feed. Thedifferent microorganisms can be of different species, or they may be ofthe same species but constitute different strains within. The productmay contain multiple species and multiple strains. For example, two,three, four, five, six, and so on different microorganisms and/orstrains can be applied. The application of multiple types of differentmicroorganisms and/or different strains lead to additive or morepreferably super additive or more preferably synergistic effects inmaintaining or improving animal health or decreasing or eliminating thepresence of pathogenic bacteria.

The amount of microorganism administered to the animal feed can be anyamount sufficient to achieve the desired increase in animal efficiencyand/or animal health. This amount can be anywhere from 1 to 10¹³organisms per kg of animal feed. For example, amounts of about 10⁴cfu/gram feed, about 5×10⁴ cfu/gram feed, about 10⁵ cfu/gram feed, about5×10⁵ cfu/gram feed, or ranges between 1 to 10¹³ organisms per kg ofanimal feed can be used. In some embodiments, the dried biological maybe administered to an animal through a variety of means including, butnot limited to, being distributed in an aqueous solution andsubsequently being applied to animal feed, water source, or directly fedto the animal, or through direct application of the product onto animalfeed or direct administration or consumption by the animal.

In certain embodiments, the microorganisms and methods of the presentdisclosure involve two or more probiotic bacteria, or in specificembodiments lactic acid-producing bacteria, for the reduction orprevention of scours in calves or for therapeutic benefit to otheranimals. These compositions would be provided in a combined amounteffective to achieve the desired effect, for example, the killing orgrowth inhibition of a pathogenic microorganism. This process mayinvolve administering different strains or species of lactic acidproducing microorganisms at the same time. In certain embodiments thedifferent strains or species may be combined into a single formulationfor administration. In other embodiments, the different strains orspecies of lactic acid producing microorganisms may be each in a singleformulation for administration. Still in other embodiments, some lacticacid producing microorganism strains or species may be combined into asingle formulation and others may be combined into a differentformulation. When more than one formulation is used the formulations maybe administered to the animal at the same time or subsequent to eachother.

In such instances, it is contemplated that one may administer bothformulations within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for administration significantly,however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4,5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, for example a formulationcontaining two species of lactic acid producing microorganisms is “A”and a second formulation containing three species of lactic acidproducing microorganisms is “B”:

In such embodiments, the administration may be, for example as such:A/B/A, B/A/B, B/B/A, A/B/B, A/B/B, B/A/A, A/B/B/B, B/A/B/B, B/B/B/A,B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, A/A/A/B,B/A/A/A, A/B/A/A or A/A/B/A. It is further contemplated that otheradministrations may be used with three or more different formulations oflactic acid producing microorganisms.

In one embodiment, the novel composition is designed for continual orperiodic administration to ruminal fermentors throughout the feedingperiod in order to reduce the incidence and severity of diarrhea and/oroverall health. In this embodiment, the composition comprises a ofmixture probiotic microorganisms supplemented with prebiotic substancesthat can be introduced into the rumen and intestines of a ruminalfermentor. The probiotic microorganisms comprising this embodiment willbe those that lead to optimal fermentation and volatile fatty acidproduction within the gastrointestinal tract of a given ruminalfermentor.

In another embodiment, the novel composition is designed for continualor periodic administration to cecal fermentors throughout the feedingperiod in order to reduce the incidence and severity of diarrhea and/oroverall health. In this embodiment, the composition comprises a mixtureof probiotic microorganisms supplemented with prebiotic substances thatcan be introduced into the cecum and intestines of a cecal fermentor.The probiotic microorganisms comprising this embodiment will be thosethat lead to optimal fermentation and volatile fatty acid productionwithin the gastrointestinal tract of a given cecal fermentor.

In yet another embodiment, the novel composition is designed forcontinual or periodic administration to intestinal fermentors throughoutthe feeding period in order to reduce the incidence and severity ofdiarrhea and/or overall health. In this embodiment, the compositioncomprises a mixture of probiotic microorganisms supplemented withprebiotic substances that can be introduced into the intestines of anintestinal fermentor. The probiotic microorganisms comprising thisembodiment will be those that lead to optimal fermentation and volatilefatty acid production within the gastrointestinal tract of a givenintestinal fermentor.

A wide range of pathogenic bacteria can potentially be inhibited oreliminated through the use of a combination of beneficial probioticbacteria or microorganisms such as lactic acid producing probioticbacteria. Specific examples of infectious diseases or conditions ofanimals which can be caused by pathogenic bacteria include, but are notlimited to: staphylococcal infections (caused, for example, byStaphylococcus aureus, Staphylococcus epidermis, or Staphylococcussaprophyticus), streptococcal infections (caused, for example, byStreptococcus pyogenes, Streptococcus pneumoniae, or Streptococcusagalactiae), enterococcal infections (caused, for example, byEnterococcus faecalis) diphtheria (caused, for example, byCorynebacterium diptheriae), anthrax (caused, for example, by Bacillusanthracis), listeriosis (caused, for example, by Listeriamonocytogenes), gangrene (caused, for example, by Clostridiumperfringens), tetanus (caused, for example, by Clostridium tetanus),botulism (caused, for example, by Clostridium botulinum), toxicenterocolitis (caused, for example, by Clostridium difficile), bacterialmeningitis (caused, for example, by Neisseria meningitidis), bacteremia(caused, for example, by Neisseria gonorrhoeae), E. coli infections(colibacilliocis), including urinary tract infections and intestinalinfections, shigellosis (caused, for example, by Shigella species),salmonellosis (caused, for example, by Salmonella species), Yersiniainfections (caused, for example, by Yersinia pestis, Yersiniapseudotuberculosis, or Yersinia enterocolitica), cholera (caused, forexample, by Vibrio cholerae), campylobacteriosis (caused, for example,by Campylobacter jejuni or Campylobacter fetus), gastritis (caused, forexample, by Helicobacter pylori), pseudomonas infections (caused, forexample, by Pseudomonas aeruginosa or Pseudomonas mallei), Haemophilusinfluenzae type B (HIB) meningitis, HIB acute epiglottitis, or HIBcellulitis (caused, for example, by Haemophilus influenzae), pertussis(caused, for example, by Bordetella pertussis), mycoplasma pneumonia(caused, for example, by Mycoplasma pneumoniae), nongonococcalurethritis (caused, for example, by Ureaplasma urealyticum),legionellosis (caused, for example, by Legionella pneumophila),syphillis (caused, for example, by Treponema pallidum), leptospirosis(caused, for example, by Leptospira interrogans), Lyme borreliosis(caused, for example, by Borrelia burgdorferi), tuberculosis (caused,for example, by Mycobacterium tuberculosis), leprosy (caused, forexample, by Mycobacterium leprae), actinomycosis (caused, for example,by Actinomyces species), nocardiosis (caused, for example, by Nocardiaspecies), chlamydia (caused, for example, by Chlamydia psittaci,Chlamydia trachomatis, or Chlamydia pneumoniae), Rickettsial diseases,including spotted fever (caused, for example, by Rickettsia ricketsii)and Rickettsial pox (caused, for example, by Rickettsia akari), typhus(caused, for example, by Rickettsia prowazekii), brucellosis (caused,for example, by Brucella abortus, Brucella melitens, or Brucella suis),and tularemia (caused, for example, by Francisella tularensis). Diseaseswith similar origins and symptoms are also known to affect animals.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit or scope of thedisclosure. The following Examples are offered by way of illustrationand not by way of limitation.

Example 1 Preparation of Reagents

a. Lactobacilli MRS Agar and Broth

Lactobacilli MRS agar and broth are recommended for the use in theisolation of Lactobacillus species. Lactobacilli MRS agar and broth arebased on the formulations of de Man et al., J. Appl. Bacteriol., 23:130,1960. Difco™ & BBL™ Manual, 2nd Edition. The agar and broth weredemonstrated by de Man et al., to support Lactobacilli growth from oral,fecal dairy and other sources. Lactobacilli MRS Agar and broth containpeptone and dextrose, both of which supply nitrogen, carbon and otherelements necessary for growth. Polysorbate 80, acetate, magnesium andmanganese provide growth factors for culturing a variety oflactobacilli.

In brief, to generate Lactobacilli MRS Agar, into one liter of distilledwater: 10.0 g proteose peptone No. 3, 10.0 g beef extract, 5.0 g yeastextract, 20.0 g dextrose, 1.0 g polysorbate 80, 2.0 g ammonium citrate,5.0 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate,2.0 g dipotassium phosphate and 15.0 g agar. Lactobacilli MRS broth isgenerated by the same methods without the addition of agar. Thesematerials are readily obtained from Becton Dickinson and Company,Franklin Lakes N.J.

b. Lactobacilli fermentation medium

Lactobacilli fermentation medium may be made by adding into 450 ml ofdistilled water the following ingredients: 4.0 g trypticase, 3.0 gcasamino acids, 6.0 g yeast extract, 0.5 g sodium acetate trihydrate,1.0 g ammonium citrate, 1.0 g potassium phosphate, 1.0 g magnesiumsulfate, 0.05 g manganese sulfate and 500 μl polyoxyethylene (20)sorbitan monooleate.

c. LBS (Lactobacillus selection) Medium

LBS medium may be made by adding into 1L of distilled water thefollowing ingredients: 10.0 g trypticase, 5.0 g yeast extract, 25.0 gsodium acetate hydrate, 20.0 g glucose, 2.0 g ammonium citrate, 6.0 gmonopotassium phosphate, 0.575 g magnesium sulfate anhydrous, 0.12 gmanganese sulfate monohydrate, 0.034 g ferrous sulfate, and 1 mlpolyoxyethylene (20) sorbitan monooleate. LBS agar may be prepared byadding 15 g of agar to 1L of the LBS medium.

d. Luria-Bertani Medium

Luria-Bertani (LB) medium may be made by adding into 1L of distilledwater the following ingredients: 10.0 g trypticase, 5.0 g yeast extract,and 10.0 g sodium chloride. LB agar may be prepared by adding 15 g ofagar to 1L of the LB medium.

Example 2

a. Growth Inhibition of E. coli by Lactic Acid Producing Bacteria

Tubes containing an MRS medium were inoculated with approximately 1×10⁴viable cells (based culture optical density) of a selected strain of E.coli and 1×10⁴ viable cells (based culture optical density) of thechallenging lactic acid bacterium strain. The following species ofprobiotic lactic acid producing bacteria were used: Lactobacillusagilis, Lactobacillus agilis, Lactobacillus delbrueckii, Lactobacillusmurinus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillussalivarius and Pediococcus acidilactici.

E. coli strain 1 was a lab isolate known as CKYL1 and strain 2,available from ATCC was 25922. The third strain was 0157:H7. Tubes werethen placed into a water bath at 37° C. and incubated for 6 hours. Afterincubation, tubes were serially diluted and 100 μl spread ontoLuria-Bertani plates to enumerate viable E. coli. The percent E. coligrowth inhibition was calculated as the difference in concentration ofviable E. coli cells in tubes co-incubated with Lactobacillus strainsfrom the control E. coli viable concentration divided by theconcentration of viable E. coli incubated without Lactobacillusmultiplied by 100. Results are shown in Table 1 and FIG. 1 forinhibition of lab strain CKYL1, Table 2 and FIG. 2 for inhibition of labstrain 25922 and Table 3 and FIG. 3 for inhibition of E. coli strain0157:H7.

TABLE 1 Growth inhibition of E. coli strain CKYL1 by Lactic AcidProducing Bacteria LAB Species Percent E. coli Growth Reduced Ranking L.agilis 81.10% 3 L. agilis 75.40% 4 L. delbrueckii 12.00% 6 L. murinus87.60% 2 L. plantarum 0.00% 7 L. reuteri 60.50% 5 L. salivarius 93.20% 1P. acidilactici −39.85% 8

TABLE 2 Growth inhibition of E. coli strain 25922 by Lactic AcidProducing Bacteria LAB Species Percent E. coli Growth Reduced Ranking L.agilis 84.83% 2 L. animalis 82.94% 4 (t) L. murinus 82.46% 6 L.plantarum 82.94% 4 (t) L. reuteri 86.26% 1 L. salivarius 84.36% 3 P.acidilactici 52.76% 8

TABLE 3 Growth inhibition of E. coli strain O157:H7 by Lactic AcidProducing Bacteria LAB Species Percent E. coli Growth Reduced Ranking L.agilis 72.88% 1 L. animalis 41.24% 4 L. murinus 64.95% 2 L. salivarius62.71% 3

b. Growth inhibition of Salmonella by Lactic Acid Producing Bacteria

Tubes containing an MRS medium were inoculated with approximately 1×10⁴viable cells (based culture optical density) of a selected species ofSalmonella and 1×10⁴ viable cells (based culture optical density) of thechallenging lactic acid bacterium strain. The following species ofprobiotic lactic acid producing bacteria were used: Lactobacillusagilis, Lactobacillus agilis, Lactobacillus delbrueckii, Lactobacillusmurinus, Lactobacillus plantarum, Lactobacillus reuteri andLactobacillus salivarius.

Two different species of Salmonella were used, Salmonella cholerasuisand Salmonella typhimurium. Tubes were then placed into a water bath at37° C. and incubated for 6 hours. After incubation, tubes were seriallydiluted and 100 μl spread onto Luria-Bertani plates to enumerate viableSalmonella. The percent Salmonella growth inhibition was calculated asthe difference in concentration of viable Salmonella cells in tubesco-incubated with Lactobacillus strains from the control Salmonellaviable concentration divided by the concentration of viable Salmonellaincubated without Lactobacillus multiplied by 100. Results are shown inTable 4 and FIG. 4 for inhibition of Salmonella cholerasuis and Table 5and FIG. 5 for inhibition of Salmonella typhimurium.

TABLE 4 Growth inhibition of Salmonella cholerasuis by Lactic AcidProducing Bacteria Percent Salmonella LAB Species Growth Reduced RankingL. agilis 67.61% 3 L. animalis 59.00% 4 L. delbrueckii 20.85% 7 L.murinus 73.80% 1 L. plantarum 44.30% 5 L. reuteri 37.50% 6 L. salivarius69.70% 2

TABLE 5 Growth inhibition of Salmonella typhimurium by Lactic AcidProducing Bacteria Percent Salmonella LAB Species Growth Reduced RankingL. agilis 85.54% 1 L. animalis 42.17% 3 L. murinus 56.60% 2 L. plantarum−18.88% 6 L. reuteri 29.00% 5 L. salivarius 20.40% 4

c. Discussion

As shown in Tables 6 and 7 below, the multiple species of lactic acidproducing bacteria inhibited the selected strains or species ofpathogenic bacteria (E. coli and Salmonella) at different levels ofeffectiveness. Thus combining multiple strains of lactic acid producingbacteria may be advisable to provide greater protection and healthbenefits to animals. Table 6 demonstrates the percentage of inhibitionagainst each pathogenic bacteria studied by selected lactic acidproducing bacteria. Likewise, Table 7 demonstrates the relative rankingof the effectiveness of inhibition against each pathogenic bacteria byselected lactic acid producing bacteria.

TABLE 6 Percentage of Inhibition Against Each Pathogenic BacteriaStudied by Selected Lactic Acid Producing Bacteria Pathogen LAB StrainE. coli (1) E. coli (2) E. coli O157:H7 S. cholerasuis S. typhimurium L.agilis 81.10% 84.83% 72.88% 67.61% 85.54% L. animalis 75.40% 82.94%41.24% 59.00% 42.17% L. delbrueckii 12.00% 20.85% L. murinus 87.60%82.46% 64.95% 73.80% 56.60% L. plantarum 0.00% 82.94% 44.30% −18.88% L.reuteri 60.50% 86.26% 37.50% 29.00% L. salivarius 93.20% 84.36% 62.71%69.70% 20.40% P. acidilactici −39.85% 52.76%

TABLE 7 Ranking of Effectiveness of Inhibition Against PathogenicBacteria by Selected Lactic Acid Producing Bacteria Pathogen LAB StrainE. coli (1) E. coli (2) E. coli O157:H7 S. cholerasuis S. typhimurium L.agilis 3 2 1 3 1 L. animalis 4 4 (t) 4 4 3 L. delbrueckii 6 7 L. murinus2 6 2 1 2 L. plantarum 7 4 (t) 5 6 L. reuteri 5 1 6 4 L. salivarius 1 33 2 5 P. acidilactici 8 8

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1. A once-weekly fed feed additive for ruminant animals that inhibitsgrowth of pathogenic bacteria in the post-ruminal digestive tract of theanimal, wherein the feed additive comprises a prepared compositionhaving 1×10¹³ cfu of Lactobacillus salivarius probiotic microorganismsper kilogram of feed which reduces viable cells of Escherichia coli at aconcentration of 1×10⁴ by at least 62.71 percent.
 2. The feed additiveof claim 1, wherein the prepared composition having 1×10¹³ cfu ofLactobacillus salivarius probiotic microorganisms per kilogram of feedreduces viable cells of Salmonella at a concentration of 1×10⁴ by atleast 42.71 percent.
 3. A once-weekly fed feed additive for ruminantanimals that inhibits growth of pathogenic bacteria in the post-ruminaldigestive tract of the animal, wherein the feed additive comprises aprepared composition having 1×10¹³ cfu of Lactobacillus agilis probioticmicroorganisms per kilogram of feed which reduces viable cells ofEscherichia coli at a concentration of 1×10⁴ by at least 72.88 percent.4. The feed additive of claim 3, wherein the prepared composition having1×10¹³ cfu of Lactobacillus agilis probiotic microorganisms per kilogramof feed reduces viable cells of Salmonella at a concentration of 1×10⁴by at least 67.61 percent.
 5. A once-weekly fed feed additive forruminant animals that inhibits growth of pathogenic bacteria in thepost-ruminal digestive tract of the animal, wherein the feed additivecomprises a prepared composition having 1×10¹³ cfu of Lactobacillusreuteri probiotic microorganisms per kilogram of feed which reducesviable cells of Escherichia coli at a concentration of 1×10⁴ by at least60.05 percent.
 6. The feed additive of claim 5, wherein the preparedcomposition having 1×10¹³ cfu of Lactobacillus reuteri probioticmicroorganisms per kilogram of feed reduces viable cells of Salmonellaat a concentration of 1×10⁴ by at least 42.17 percent.